Use of a saccharomyces cerevisiae engineered strain in the preparation of squalene
Patent Information
- Application Number
- CN202311198223.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-18
- Publication Date
- 2026-10-30
- Estimated Expiration
- 2043-09-18
AI Technical Summary
然而，目前针对角鲨烯合成途径基因靶点的改造已出现边际递减效应，急需在新的维度上寻找有效改造靶点，以进一步提高角鲨烯产量
[0019]本发明对酵母细胞进行针对LncRNA靶点的改造，改造后的细胞用于表达截短型HMG-CoA还原酶1，得到高产角鲨烯的工程菌，相比未经LncRNA靶点而用于表达截短型HMG-CoA还原酶1的对照菌株L1T，角鲨烯的产量均超过211.2％。其中，L3T菌株产量可达35.16mg/L，相比对照菌株L1T角鲨烯的产量(15.16mg/L)提高了231.8％以上。不同于提高重组tHMG1表达质粒拷贝数等传统方法，该酵母工程菌仅从出发菌底盘细胞自身出发，围绕LncRNA相关靶点的敲除而展开。本发明创新性地改造长非编码RNA靶点，成功发现了酿酒酵母中有效提升角鲨烯产量的长非编码RNA靶点。酵母工程菌用于提高其他高经济价值内源酶有较强的潜力，具备广阔的应用前景和重要的实践意义。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to the application of an engineered strain of Saccharomyces cerevisiae in the preparation of squalene. Background Technology
[0002] Squalene is a linear triterpenoid compound widely found in plants, animals, and humans, possessing various beneficial properties such as antioxidant and antitumor effects. Squalene was initially isolated from shark liver oil. The increasing market demand for squalene may lead to overfishing and other environmental problems. Although recent studies have found that high-purity squalene can be obtained through plant extraction, the low squalene content in plants, the cumbersome extraction process, and the susceptibility of raw materials to seasonal and geographical influences all contribute to the high price of the extracted product. Therefore, plant extraction is not suitable for large-scale industrial production and commercial promotion of squalene.
[0003] Saccharomyces cerevisiae is gradually becoming an effective host for the production of various terpenoids, including squalene. Using Saccharomyces cerevisiae for squalene synthesis has several advantages: simple culture conditions, low cost, short production cycle, and relatively simple downstream product separation. Therefore, the fermentation synthesis of terpenoids using Saccharomyces cerevisiae has been widely applied. The mevalonate pathway (MVA) in Saccharomyces cerevisiae is responsible for providing precursors for terpenoid synthesis. Overexpression of the enzyme genes HMG1 and IDI1, which are rate-limiting steps in the mevalonate (MVA) pathway, can increase squalene yield; inhibiting the expression of ERG1 or ERG7 to reduce the downward metabolism of squalene can reduce squalene consumption, thereby increasing squalene accumulation. However, current modifications targeting genes in the squalene synthesis pathway have shown diminishing marginal returns, necessitating the search for effective modification targets in new dimensions to further improve squalene yield.
[0004] Therefore, modifying yeast strains at the non-coding RNA level through genetic engineering and metabolic engineering to increase squalene production is of great significance and practical application value. Summary of the Invention
[0005] The objective of this invention is achieved through the following technical solution: the application of an engineered strain of *Saccharomyces cerevisiae* in the preparation of squalene, wherein the engineered yeast strain has the following characteristics: at least one of LncRNA SUT067, LncRNA SUT433 and LncRNA CUT782 is deleted or mutated, and contains a nucleic acid encoding HMG-CoA reductase 1; preferably, the process includes the following steps: inoculating the engineered strain of *Saccharomyces cerevisiae* into a fermentation medium and culturing it, and collecting the fermentation product to obtain squalene.
[0006] The engineered strain of brewer's yeast is preferably obtained through the following steps:
[0007] (1) Modification of the chassis cells of the starting strain of the engineered Saccharomyces cerevisiae: At least one of LncRNA SUT067, LncRNA SUT433 and LncRNA CUT782 in the starting strain was deleted or mutated to obtain a preliminarily modified strain.
[0008] (2) Construction of tHMG1 expression recombinant vector: The nucleic acid encoding HMG-CoA reductase 1 was constructed on the expression vector to obtain the tHMG1 expression recombinant vector;
[0009] (3) Obtaining the engineered strain of Saccharomyces cerevisiae: The tHMG1 expression recombinant vector was transformed into the preliminarily modified strain obtained in step (1) to obtain the engineered strain of Saccharomyces cerevisiae.
[0010] The starting strain of the engineered Saccharomyces cerevisiae is preferably a strain with a CEN.PK background. This strain is a commonly used yeast strain and is widely used in metabolic engineering and systems biology research in industry and academia (Microb CellFact, 2012, 11, 36), with good representativeness and universality; preferably Saccharomyces cerevisiae CEN.PK 113-5D.
[0011] The deletion is preferably removed without scarring by homologous recombination or CRISPR / Cas9 gene editing technology.
[0012] The mutation described above renders the corresponding LncRNA ineffective, and can be obtained through homologous recombination or CRISPR / Cas9 gene editing technology.
[0013] The tHMG1 mentioned is HMG-CoA reductase 1; preferably, it is a truncated HMG-CoA reductase 1 derived from Saccharomyces cerevisiae.
[0014] The preferred sequence encoding the nucleic acid is shown in SEQ ID No. 1.
[0015] The preferred expression vectors are p416GAL, p416GPD, p416TEF, p416ADH, p416CYC1, p426GPD, p426TEF, p426ADH, or p426CYC1.
[0016] The preferred fermentation medium is YPG medium.
[0017] The preferred culture conditions are 28–32°C and 150–250 rpm for 80–120 h; more preferably, 30°C and 200 rpm for 96 h.
[0018] The present invention has the following advantages and effects compared with the prior art:
[0019] This invention modifies yeast cells to target lncRNAs. The modified cells are used to express truncated HMG-CoA reductase 1, resulting in a high-squalene-producing engineered strain. Compared to the control strain L1T, which was used to express truncated HMG-CoA reductase 1 without lncRNA targeting, the squalene yield exceeded 211.2%. Specifically, the L3T strain achieved a yield of 35.16 mg / L, representing a 231.8% increase compared to the control strain L1T (15.16 mg / L). Unlike traditional methods such as increasing the copy number of recombinant tHMG1 expression plasmids, this engineered yeast strain focuses solely on the knockout of lncRNA-related targets within the starting cell chassis. This invention innovatively modifies long non-coding RNA targets, successfully discovering long non-coding RNA targets in Saccharomyces cerevisiae that effectively enhance squalene production. Engineered yeast strains have strong potential for increasing the production of other high-economic-value endogenous enzymes, possessing broad application prospects and significant practical implications. Attached Figure Description
[0020] Figure 1 This is a schematic diagram of the composition of the recombinant expression plasmid p416-Pgal7-tHMG1-Tcyc1 carrying the URA3 selection marker.
[0021] Figure 2 This is an agarose gel electrophoresis image of the recombinant expression plasmid p416-Pgal7-tHMG1-Tcyc1 verified by restriction endonucleases XbaI and BamHI; lane M is the DNA molecular weight marker.
[0022] Figure 3 This is an agarose gel electrophoresis image of PCR identification of the knockout strains SUT067, SUT433, and CUT782, where lane M is the DNA molecular weight marker.
[0023] Figure 4 These are agarose gel electrophoresis images; (A) is an agarose gel electrophoresis image of PCR identification of the empty p416-Pgal7-Tcyc1 plasmid transformed into strain CEN.PK 113-5D; (B) is an agarose gel electrophoresis image of PCR identification of the successful transformation of the p416-Pgal7-tHMG1-Tcyc1 plasmid into strains ΔSUT067, ΔSUT433, and ΔCUT782; in the images, lane M is the DNA molecular weight marker, EP is the empty p416-Pgal7-Tcyc1 plasmid, and p416-tHMG1 is the p416-Pgal7-tHMG1-Tcyc1 plasmid.
[0024] Figure 5This is a graph showing the yield of squalene synthesized by in vitro fermentation of the L0T, L1T, L2T, L3T and L4T yeast engineered strains in Example 4. Detailed Implementation
[0025] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.
[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0027] Unless otherwise specified, experimental methods in the following examples are generally performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (Beijing: Science Press, 2017) and Yeast Genetics: A Laboratory Manual (Beijing: Science Press, 2016).
[0028] The CRISPR technology used in the following examples is described in the prior art (Zhang et al. A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomycescerevisiae. Nat Commun 2019, 10(1):1053). Furthermore, the sequences of the psgtRNA, pCas, and pScURA plasmid fragments used in the following examples are disclosed in the appendix to the document "Zhang et al. A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomycescerevisiae. Nat Commun 2019, 10(1):1053". The pScURA plasmid fragment can be obtained through artificial synthesis.
[0029] To better understand the content of this invention, the background strain of Saccharomyces cerevisiae CEN.PK 113-5D (which can be obtained from EUROSCARF) is used as the starting strain for further explanation of specific embodiments.
[0030] The culture media involved in the following examples are as follows:
[0031] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is deionized water; solid medium is supplemented with 2% agar powder.
[0032] LB / AMP medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is deionized water; solid medium with 2% agar powder added, after sterilization, cool to about 40°C, add 100 μg / mL ampicillin (filter sterilization).
[0033] YPD medium: 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose (sterilized separately and then added), solvent is deionized water; solid medium is supplemented with 2% agar powder.
[0034] SC-Ura auxotrophic medium: 0.77 g / L CSM-Ura, 1.7 g / L YNB w / o AA & w / o (NH4)2SO4 (Yeast Nitrogen Base without Amino acids and without Ammonium sulphate), 5.0 g / L (NH4)2SO4, 20 g / L glucose (Note: glucose is sterilized separately), adjust pH to 5.5-6.0; add 2% agar powder to solid medium; solvent is deionized water.
[0035] The methods involved in the following embodiments are as follows:
[0036] Plasmid construction:
[0037] (1) Gibson assembly method, the specific operation shall be carried out in accordance with the NEB Gibson Assembly Cloning kit (part number E2611, NEB) instruction manual;
[0038] (2) Golden gate assembly method, specific operation according to Follow the instructions for use of the Golden Gate Assembly Kit (Catalog No. E1602L, NEB);
[0039] (3) Transform 5 μL of the assembly system into 50 μL of E. coli DH5α competent cells, spread them on LB / AMP solid medium and culture overnight;
[0040] (4) Positive clones were obtained by screening, and plasmids were extracted after amplification and culture. The specific extraction process was carried out in accordance with the instructions of HiPure Plasmid Micro Kit (product number P1001-03, Magen).
[0041] Yeast strain transformation:
[0042] Unless otherwise specified, the lithium acetate conversion method shall be used. For specific operation procedures, please refer to the relevant standards and specifications.
[0043] Recombinant Saccharomyces cerevisiae strain OD600nm Detection methods:
[0044] A single colony of *Saccharomyces cerevisiae* was inoculated into 3 mL of fermentation medium and cultured on a shaker at 30°C and 200 rpm. The fermentation broth was then diluted appropriately and its OD (exponential growth factor) was measured using a UV spectrophotometer. 600nm .
[0045] Squalene detection method:
[0046] Squalene standards were purchased from Macklin (Shanghai, China). The content was determined using Shimadzu high-performance liquid chromatography with a Hypers11 ODS C18 column (250×4.6mm), a detection wavelength of 210nm, a flow rate of 1mL / min, a column temperature of 40℃, and elution with 100% acetonitrile for 20min.
[0047] The primer sequences involved in the following examples are shown in Table 1:
[0048] Table 1: Primer sequences (5'-3')
[0049] Primer-A1R GAGCTCCAGCTTTTGTTCCCT Primer-A2F AAAAGCTGGAGCTCTTTGCCAGCTTACTATCCTTCTTGA Primer-A2R TCTTCACCAATTGGTCCATTTTTGAGGGAATATTCAACTGTTTTT Primer-A3F AAAAACAGTTGAATATTCCCTCAAAAATGGACCAATTGGTGAAGA Primer-A3R ACTAATTACATGAGGATCCTTAGGATTTAATGCAGGTGACGGACCCA Primer-A4F AAAGGTCTCAGATCCTATCGCCTCTTGGTTTTTAGTTTTAGAGCTAGAAATAGCAAGT Primer-A4R AAAGGTCTCA TGCGCAAGCCCGGAATCGAACCGGG Primer-A5F AAAGGTCTCACGCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC Primer-A5R AAAGGTCTCAAAACCTAGACACAGGGTAATAACTGATATAATTAAATTGAAGCTC Primer-A6F GATATATAGCCATAATGAATCAACAAATTGTATTATTATTTCCGACAACAGCATTGCCA Primer-A6R GAAGTCTCTTTGGAAAAACCAACCTTGATCGCGCAGCAATGTGGCAATGCTGTTGTCGG Primer-A7F GACCAGGTTCTACTCTTCAC Primer-A7R GCTCTTCTACAGGCTCAAC Primer-A8F GCCAGCTTACTATCCTTCTT Primer-A8R TAATGTTACATGCGTACACG Primer-A9F AAAGGTCTCAGATCCGATAATGTGCTAGCAAAGG GTTTTAGAGCTAGAAATAGCAAGT Primer-A10F GAAGGAAGAACGTTATGTTATTAATGGACTTTTAGTGTCATCGAATTTCAGATTGATTT Primer-A10R TCGTTTTTTGCCTCTTCTTATGTCATAAGAACTTATGTGAAAAATCAATCTGAAATTCG Primer-A11F GTAGTCGCAGAGACAACAT Primer-A11R AAATGGCACCAAACTCCC Primer-A12F AAAGGTCTCAGATCCAGGTGACGATGAAGTAGTAAGTTTTAGAGCTAGAAATAGCAAGT Primer-A13F ATATGAGGTTAGGGAAGCCGGATAAAGAAAATAGAGTACTTTAAAACTGTGCAATTTTG Primer-A13R AAGGAAACGTACTAAGCTCAATTTGTCCACAGTATAATCAACAAAATTGCACAGTTTTA Primer-A14F CTACAGAGAGCCGTGAGAGTGAG Primer-A14R GATGCGAGAATACGAGATTA
[0050] Example 1: Obtaining the gene encoding HMG-CoA reductase 1 in Saccharomyces cerevisiae
[0051] Based on the nucleotide sequence of HMG-CoA reductase 1 (P12683) in Saccharomyces cerevisiae published in the UniProt database, the DNA coding sequence of HMG-CoA reductase 1, from 1591 to 3165 bp (1575 bp in total), was cloned using the genome of Saccharomyces cerevisiae CEN.PK 113-5D as a template. The start codon ATG (as shown in SEQ ID No. 1) was added to the truncated N-terminus.
[0052] Example 2: Construction of the expression vector for HMG-CoA reductase 1 in Saccharomyces cerevisiae
[0053] The structure of the truncated HMG-CoA reductase 1 expression vector p416-Pgal7-tHMG1-Tcyc1 from Saccharomyces cerevisiae is as follows: Figure 1 As shown, the specific construction process is as follows:
[0054] (1) Primers Primer-A1F / Primer-A1R were used to amplify the plasmid framework using p416-GPD plasmid as a template; PCR amplification included routine denaturation, annealing and extension steps (the same below); the annealing temperature was 56℃, the extension temperature was 72℃ for 300s, and 30 cycles of amplification were performed.
[0055] (2) Using the primer pair Primer-A2F / Primer-A2R, the GAL7 promoter fragment with homologous arms was amplified using the genome of Saccharomyces cerevisiae CEN.PK 113-5D as a template; the annealing temperature of PCR amplification was 56℃, the extension temperature was 72℃ for 20s, and 30 cycles were performed.
[0056] (3) Using the primer pair Primer-A3F / Primer-A3R, the tHMG1 fragment with homologous arms was amplified using the genome of Saccharomyces cerevisiae CEN.PK 113-5D as a template; the annealing temperature of PCR amplification was 54℃, the extension temperature was 72℃ for 20s, and 30 cycles were performed.
[0057] (4) Using Gibson assembly technology, the above three fragments are spliced together and transformed into Escherichia coli DH5α.
[0058] Sequencing and enzyme digestion (Agarose gel electrophoresis image after XbaI and BamHI digestion, as shown below) Figure 2 As shown in the figure, the plasmid construction was successfully verified and named p416-Pgal7-tHMG1-Tcyc1.
[0059] Example 3: Construction of an engineered Saccharomyces cerevisiae strain expressing tHMG-CoA reductase 1
[0060] 3.1 Knockout of SUT067 to achieve high squalene production
[0061] The non-coding RNA SUT067 knockout process is as follows:
[0062] (1) Construction of plasmid pCas9-SUT067
[0063] The nucleotide sequence of the non-coding RNA SUT067 is shown in SEQ ID NO.2.
[0064] Using plasmid psgtRNA as a template, a gRNA sequence targeting SUT067 was obtained by amplification with primer pair Primer-A4F / Primer-A4R. The annealing temperature of PCR amplification was 56℃, followed by extension at 72℃ for 20s, and 30 amplification cycles were performed.
[0065] Using the pScURA plasmid fragment as a template, a 1127bp plasmid framework fragment with the nutrient marker URA3 was amplified by primer pair Primer-A5F / Primer-A5R; the annealing temperature for PCR amplification was 56℃, followed by extension at 72℃ for 30s, and 30 amplification cycles were performed.
[0066] Using the Golden Gate assembly technique, the two fragments mentioned above were spliced together with a pCas plasmid containing a Cas9 plasmid fragment, and the resulting plasmid was named pCas9-SUT067.
[0067] (2) PCR amplification of the SUT067 repair fragment
[0068] A 59bp primer pair, Primer-A6F / Primer-A6R, that perfectly matched the upstream and downstream of SUT067 was synthesized. The targeted repair fragment with 50bp homologous arms to the upstream and downstream of SUT067 was obtained by PCR amplification and gel recovery. The annealing temperature of PCR amplification was 56℃, followed by extension at 72℃ for 20s, and 30 cycles were performed.
[0069] (3) The plasmid pCas9-SUT067, along with the SUT067 repair fragment, was transformed into strain CEN.PK 113-5D using the lithium acetate transformation method. The transformed strains were plated on SC-Ura solid culture media and incubated at 30℃ for 3–4 days. Single-colony PCR was performed using primer pairs Primer-A7F / Primer-A7R to verify the transformations and screen for positive transformants (results are shown in Figure 1). Figure 3 As shown in the figure, plasmid pCas9-SUT067 was removed, and the resulting Saccharomyces cerevisiae strain was named L2.
[0070] Transform plasmid p416-Pgal7-tHMG1-Tcyc1 into strain L2 to express truncated HMG-CoA reductase 1:
[0071] Saccharomyces cerevisiae L2 was prepared into competent cells, and the truncated HMG-CoA reductase 1 expression plasmid p416-Pgal7-tHMG1-Tcyc1 was transformed into these competent cells using the lithium acetate conversion method. The cells were then plated onto SC-Ura solid medium and cultured at 30°C for 3-4 days. Single-colony PCR was performed using primers Primer-A8F / Primer-A8R to verify the colonies and screen for positive strains (results are shown in the figure). Figure 4 As shown in (B) in the figure, it is named Saccharomyces cerevisiae L2T.
[0072] 3.2 Knockout of SUT433 to achieve high squalene production
[0073] The non-coding RNA SUT433 knockout process is as follows:
[0074] (1) Constructing plasmid pCas9-SUT433
[0075] The nucleotide sequence of the non-coding RNA SUT433 is shown in SEQ ID NO.3.
[0076] Using plasmid psgtRNA as a template, a gRNA sequence targeting SUT433 was obtained by amplification with primer pair Primer-A9F / Primer-A4R. The annealing temperature of PCR amplification was 56℃, extension at 72℃ for 20s, and amplification was performed for 30 cycles.
[0077] Using the pScURA plasmid fragment as a template, a 1127bp plasmid framework fragment with the nutrient marker URA3 was amplified by primer pair Primer-A5F / Primer-A5R. The annealing temperature for PCR amplification was 56℃, the extension temperature was 72℃ for 30s, and the amplification was performed for 30 cycles.
[0078] Using the Golden Gate assembly technique, the two fragments mentioned above were spliced together with a pCas plasmid containing a Cas9 plasmid fragment, and the resulting plasmid was named pCas9-SUT433.
[0079] (2) PCR amplification of SUT433 repair fragment
[0080] A 59bp primer pair, Primer-A10F / Primer-A10R, that was perfectly matched to the upstream and downstream of SUT433 was synthesized. The targeted repair fragment with 50bp homologous arms to the upstream and downstream of SUT433 was obtained by PCR amplification and gel recovery. The annealing temperature of PCR amplification was 56℃, extension temperature was 72℃ for 20s, and amplification was performed for 30 cycles.
[0081] (3) The plasmid pCas9-SUT433, along with the SUT433 repair fragment, was transformed into strain CEN.PK 113-5D using the lithium acetate transformation method. The transformed strain was plated on an SC-Ura solid culture medium and incubated at 30℃ for 3-4 days. Single-colony PCR was performed using primer pairs Primer-A11F / Primer-A11R to verify the transformations and screen for positive transformants (results are shown in the figure). Figure 3 As shown in the figure, plasmid pCas9-SUT433 was removed, and the resulting Saccharomyces cerevisiae strain was named L3.
[0082] Transform plasmid p416-Pgal7-tHMG1-Tcyc1 into strain L3 to express truncated HMG-CoA reductase 1:
[0083] Saccharomyces cerevisiae L3 was prepared as competent cells, and the truncated HMG-CoA reductase 1 expression plasmid p416-Pgal7-tHMG1-Tcyc1 of Saccharomyces cerevisiae was transformed into these competent cells using the lithium acetate conversion method. The cells were then plated on SC-Ura solid medium and cultured at 30℃ for 3-4 days. Single-colony PCR was performed using primers Primer-A8F / Primer-A8R to verify the positive strains (results are shown in the figure). Figure 4As shown in (B) in the figure, it is named Saccharomyces cerevisiae L3T.
[0084] 3.3 Knockout of CUT782 to achieve high squalene production
[0085] The non-coding RNA CUT782 knockout process is as follows:
[0086] (1) Constructing plasmid pCas9-CUT782
[0087] The nucleotide sequence of the non-coding RNA CUT782 is shown in SEQ ID NO.4.
[0088] Using plasmid psgtRNA as a template, a gRNA sequence with a 20bp target CUT782 was amplified by primer pair Primer-A12F / Primer-A4R. The annealing temperature for PCR amplification was 56℃, the extension temperature was 72℃ for 20s, and the amplification was repeated for 30 cycles.
[0089] Using the pScURA plasmid fragment as a template, a 1127bp plasmid framework fragment with the nutrient marker URA3 was amplified by primer pair Primer-A5F / Primer-A5R. The PCR amplification temperature was 56℃, extension temperature was 72℃ for 30s, and amplification was performed for 30 cycles.
[0090] Using the Golden Gate assembly technique, the two fragments mentioned above were spliced together with a pCas plasmid containing a Cas9 plasmid fragment, and the resulting plasmid was named pCas9-CUT782.
[0091] (2) PCR amplification of the CUT782 repair fragment
[0092] A 59bp primer pair, Primer-A13F / Primer-A13R, that was perfectly matched with the upstream and downstream of CUT782 was synthesized. The targeted repair fragment with 50bp homologous arms with CUT782 was obtained by PCR amplification and gel recovery. The PCR amplification annealing temperature was 56℃, extension temperature was 72℃ for 20s, and amplification was performed for 30 cycles.
[0093] (3) The plasmid pCas9-CUT782, along with the CUT782 repair fragment, was transformed into strain CEN.PK 113-5D using the lithium acetate transformation method. The transformed strains were plated on SC-Ura solid culture media and incubated at 30℃ for 3-4 days. Single-colony PCR was performed using primer pairs Primer-A14F / Primer-A14R to verify the transformations and screen for positive transformants (results are shown in the figure). Figure 3 As shown in the figure, plasmid pCas9-CUT782 was removed, and the resulting Saccharomyces cerevisiae strain was named L4.
[0094] Transform plasmid p416-Pgal7-tHMG1-Tcyc1 into strain L4 to express truncated HMG-CoA reductase 1:
[0095] Saccharomyces cerevisiae L4 was prepared as competent cells, and the truncated HMG-CoA reductase 1 expression plasmid p416-Pgal7-tHMG1-Tcyc1 of Saccharomyces cerevisiae was transformed into these competent cells via lithium acetate conversion. The cells were then plated on SC-Ura solid medium and cultured at 30°C for 3-4 days. Single-colony PCR was performed using primers Primer-A8F / Primer-A8R to verify the colonies and screen for positive strains (results are shown in the figure). Figure 4 As shown in (B) in the figure, it is named Saccharomyces cerevisiae L4T.
[0096] Example 4: Production of Squalene by Fermentation of Engineered Strains
[0097] (1) Saccharomyces cerevisiae strains L0T, L1T, and the aforementioned L2T, L3T, and L4T were inoculated from plates into 2.5 mL of YPG fermentation medium and cultured at 30℃ and 200 rpm for 96 h. L0T was used as an empty plasmid control. The preparation process of L0T was as follows: Saccharomyces cerevisiae CEN.PK 113-5D was prepared into competent cells, and the empty plasmid p416-Pgal7-Tcyc1 was transformed into the competent cells by lithium acetate conversion. The cells were then plated on SC-Ura solid medium and cultured at 30℃ for 3-4 days. Single colony PCR verification was performed using primers Primer-A8F / Primer-A8R to screen for positive strains (results are shown in Figure 1). Figure 4 (As shown in (A)), it was named *Saccharomyces cerevisiae* L0T. L1T is the control strain. The preparation process of L1T is as follows: *Saccharomyces cerevisiae* CEN.PK 113-5D was prepared into competent cells, and the *Saccharomyces cerevisiae* HMG-CoA reductase 1 expression plasmid p416-Pgal7-tHMG1-Tcyc1 was transformed into the competent cells by lithium acetate conversion. The cells were plated on SC-Ura solid medium and cultured at 30℃ for 3-4 days. Single colony PCR was performed using primers Primer-A8F / Primer-A8R to verify the positive strains (results are shown in Figure 1). Figure 4 As shown in (B) in the figure, it is named Saccharomyces cerevisiae L1T.
[0098] (2) After fermentation for 96 hours, the fermentation broth was diluted 50 times and the OD was measured using a UV spectrophotometer. 600nm .
[0099] (3) After centrifuging 1 mL of fermentation broth, take 500 μL of the supernatant and add 1000 μL of ethyl acetate.
[0100] (4) Shake to mix the mixture, centrifuge, take 500 μL of supernatant and filter.
[0101] (5) Determine the squalene yield.
[0102] The detection results of yeast expressing truncated HMG-CoA reductase 1 are as follows: Figure 5 As shown, the squalene yield (15.16 mg / L) of strains L2T, L3T, and L4T was increased by 211.2%, 231.8%, and 226.7% respectively compared with strain L1T. Figure 5 It was also shown that the squalene yield of the L1T control strain transformed with the p416-Pgal7-tHMG1-Tcyc1 plasmid was 326% higher than that of the L0T control strain with the empty plasmid p416-Pgal7-Tcyc1 (4.639 mg / L).
[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. The application of an engineered strain of *Saccharomyces cerevisiae* in the preparation of squalene, characterized in that: The yeast engineered strain has the following characteristics: at least one of LncRNA SUT067, LncRNA SUT433 and LncRNA CUT782 is deleted or mutated, and it contains the coding nucleic acid that can express HMG-CoA reductase 1; The nucleotide sequence of the LncRNA SUT067 is shown in SEQ ID NO.2; The nucleotide sequence of the LncRNA SUT433 is shown in SEQ ID NO.3; The nucleotide sequence of the LncRNA CUT782 is shown in SEQ ID NO.4; The aforementioned deletions or mutations render the corresponding LncRNAs no longer functional. The HMG-CoA reductase 1 mentioned above is a truncated form of Saccharomyces cerevisiae HMG-CoA reductase 1; The sequence of the nucleic acid encoding HMG-CoA reductase 1 is shown in SEQ ID No.
1.
2. The application according to claim 1, characterized in that... The process includes the following steps: inoculating the engineered strain of Saccharomyces cerevisiae into a fermentation medium and culturing it, and collecting the fermentation products to obtain squalene.
3. The application according to claim 1 or 2, characterized in that: The engineered strain of brewer's yeast was obtained through the following steps: (1) Modification of the chassis cells of the starting strain of the engineered Saccharomyces cerevisiae: At least one of LncRNASUT067, LncRNA SUT433 and LncRNA CUT782 in the starting strain was deleted or mutated to obtain a preliminarily modified strain. (2) Construction of tHMG1 expression recombinant vector: The nucleic acid encoding HMG-CoA reductase 1 was constructed on the expression vector to obtain the tHMG1 expression recombinant vector; (3) Obtaining the engineered strain of Saccharomyces cerevisiae: The tHMG1 expression recombinant vector was transformed into the preliminarily modified strain to obtain the engineered strain of Saccharomyces cerevisiae.
4. The application according to claim 1, characterized in that: The starting strain of the engineered Saccharomyces cerevisiae is a strain with a CEN.PK background.
5. The application according to claim 4, characterized in that: The starting strain of the engineered Saccharomyces cerevisiae strain is Saccharomyces cerevisiae CEN.PK 113-5D.
6. The application according to claim 3, characterized in that: The expression vector is p416GAL, p416GPD, p416TEF, p416ADH, p416CYC1, p426GPD, p426TEF, p426ADH or p426CYC1.
7. The application according to claim 2, characterized in that: The fermentation medium is YPG medium; The culture conditions are as follows: culture at 28–32℃ and 150–250 rpm for 80–120 h.